NEBNext Small Fragment Removal (E6116)

Protocol

Add the End Repaired/dA-Tailed/Adaptor ligated DNA sample directly to the previously prepared Ampure beads. Vortex briefly to mix, followed by a quick spin to collect liquid from the sides of the tube.

Incubate at room temperature for 5 minutes.

Place the tube on a Magnetic Separator.

When the beads have collected to the wall of the tube and the solution is clear, remove and discard the supernatant. Be careful not to disturb the beads.

Add 100 μl of TE and vortex until the beads are completely re-suspended.

Add 500 μl of NEBNext Sizing Buffer and briefly vortex to mix.

Incubate at room temperature for 5 minutes.

Place the tube on a Magnetic Separator.

When the beads are collected to the wall of the tube and the solution is clear, remove and discard the supernatant. Be careful not to disturb the beads.

Repeat steps 5-9 one time.

Keep the tube on the magnet and wash the beads twice with 1 ml of 70% ethanol.

Keep the tube on the magnet, uncapped, and let the pellet air dry until there is no visible liquid on the sides of the tube. This typically takes 5 minutes.

Remove the tube from the magnet, add 53 μl of TE, vortex to re-suspend the beads and spin briefly.

Place the tube on the magnet, when the beads are collected to the wall of the tube, transfer 50 μl of the supernatant (library), to a new 1.7 ml micro-centrifuge tube. Be careful not to transfer any beads.